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Journal of Clinical Microbiology Oct 2017Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of...
Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of viral RNA is highly sensitive and specific, but periods of viremia and viruria are brief, limiting the utility of ZIKV RNA assays. Instead, most ZIKV infections are diagnosed serologically, using an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for screening, followed by a confirmatory plaque reduction neutralization test (PRNT). Typical turnaround times vary, due to assay incubation periods and a lack of clinical laboratories performing these tests. Recently, a novel luciferase-ZIKV- and -dengue virus (DENV)-based serological assay, which considerably improves the turnaround times and throughput for ZIKV diagnosis, was described. Using the traditional PRNT as a reference method, we evaluated the performance characteristics of the reporter virus neutralization test (RVNT) with 258 clinical serum specimens. The ZIKV RVNT produced primary ZIKV screening and secondary confirmation results in 4 days, with 100% reproducibility. As a screening assay, the ZIKV RVNT displayed excellent diagnostic accuracy, sensitivity, and specificity of 98.2%, 100%, and 98.1%, respectively. As a confirmatory assay, the ZIKV RVNT titers displayed 93.1% agreement with the traditional ZIKV PRNT titers. Overall, the RVNT accurately and reliably detects neutralizing antibodies in patient serum specimens, with improved turnaround times, and can be used for the serological detection of ZIKV infections. Due to the homogeneous 96-well format, the RVNT has also significantly improved the assay throughput to allow testing of a large number of specimens in a single run.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Cell Line; Chlorocebus aethiops; Dengue; Dengue Virus; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin M; Neutralization Tests; Reproducibility of Results; Sensitivity and Specificity; Vero Cells; Viremia; Zika Virus; Zika Virus Infection
PubMed: 28768729
DOI: 10.1128/JCM.00975-17 -
Journal of Virological Methods Oct 2017Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization...
Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level 3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities. Evaluation with a broad panel of experimental sera and field sera demonstrated that this novel VNT is faster and more sensitive than the classical VNT.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Goats; Green Fluorescent Proteins; Immunoglobulin G; Neutralization Tests; Rift Valley Fever; Rift Valley fever virus; Ruminants; Sensitivity and Specificity
PubMed: 28583857
DOI: 10.1016/j.jviromet.2017.06.001 -
Viruses Mar 2020Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus in the family . The...
Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus in the family . The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.
Topics: Fluorescent Antibody Technique; Genes, Reporter; Genetic Engineering; Green Fluorescent Proteins; Neutralization Tests; Picornaviridae; Reverse Genetics
PubMed: 32150804
DOI: 10.3390/v12030283 -
PLoS Neglected Tropical Diseases Mar 2010Antibodies play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies, the Fc- and complement-dependent... (Review)
Review
Antibodies play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies, the Fc- and complement-dependent activities of these multifunctional proteins may also be critical in their ability to provide protection against most viruses. Protection against viral pathogens in vivo is complex, and while virus neutralization--the ability of antibody to inactivate virus infectivity, often measured in vitro--is important, it is often only a partial contributor in protection. The rapid fluorescent focus inhibition test (RFFIT) remains the "gold standard" assay to measure rabies virus-neutralizing antibodies. In addition to neutralization, the rabies-specific antigen-binding activity of antibodies may be measured through enzyme-linked immunosorbent assays (ELISAs), as well as other available methods. For any disease, in selecting the appropriate assay(s) to use to assess antibody titers, assay validation and how they are interpreted are important considerations-but for a fatal disease like rabies, they are of paramount importance. The innate limitations of a one-dimensional laboratory test for rabies antibody measurement, as well as the validation of the method of choice, must be carefully considered in the selection of an assay method and for the interpretation of results that might be construed as a surrogate of protection.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Enzyme-Linked Immunosorbent Assay; Humans; Neutralization Tests; Rabies; Rabies virus
PubMed: 20231877
DOI: 10.1371/journal.pntd.0000595 -
PloS One 2022There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently...
There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Chlorocebus aethiops; Fluorescence; Humans; Neutralization Tests; Vero Cells; Yellow Fever; Yellow fever virus
PubMed: 35139078
DOI: 10.1371/journal.pone.0262149 -
Journal of Clinical Microbiology Aug 2020Lumpy skin disease (LSD) is an emerging, transboundary viral pox disease affecting cattle of all ages and breeds. The serological assay for monitoring immunity following...
Lumpy skin disease (LSD) is an emerging, transboundary viral pox disease affecting cattle of all ages and breeds. The serological assay for monitoring immunity following vaccination is a virus neutralization test (VNT/OIE) that determines the neutralization index (NI). The first validated enzyme-linked immunosorbent assay (ELISA; IDVet) has become commercially available, facilitating large-scale serosurveillance for LSD. Although the VNT is labor intensive and time consuming, it is still the recommended test by the OIE. Thus, in this study, we modified the virus neutralization test by employing Madin-Darby bovine kidney (MDBK) cells. The qualitative results obtained with the modified method were compared to the qualitative results obtained by VNT/OIE and ELISA. We used blood sera received within a surveillance program for LSD in 2018. In total, 291 serum samples were tested using VNT/MDBK and ELISA. Of 291 samples, 80 samples were tested by VNT/OIE and used for comparison of the performances between VNT/MDBK and VNT/OIE. The compatibility of results obtained by VNT/MDBK and VNT/OIE resulted in a kappa index of 0.9 with overall proportion agreement of 0.96. Agreement between VNT/MDBK and VNT/OIE was achieved in 56 positive and 21 negative samples. The compatibility of results obtained by ELISA and VNT/MDBK were compared on 291 samples in total and resulted in a kappa index 0.834 with overall proportion agreement of 0.955. Agreement between ELISA and VNT/MDBK was achieved in 238 positive and 40 negative samples. The results obtained demonstrated a strong correlation between VNT/MDBK and the other two methods, indicating the suitability of VNT/MDBK for the detection of the LSD virus-specific neutralizing antibodies.
Topics: Animals; Antibodies, Viral; Cattle; Enzyme-Linked Immunosorbent Assay; Lumpy skin disease virus; Neutralization Tests; Serologic Tests
PubMed: 32434783
DOI: 10.1128/JCM.00348-20 -
Virologica Sinica Jun 2021The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody...
The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAdV-3) are the microneutralization (MN) assay, which has insufficient sensitivity, and the plaque reduction neutralization test (PRNT), which is not suitable for high-throughput screening. Herein, we describe the development of a flow cytometry-based neutralization (FCN) assay for measuring the neutralization of sera, cell culture supernatants, and chimeric antibodies against HAdV-3 on the basis of a recombinant HAdV-3 (rHAdV-3) construct expressing the enhanced green fluorescent protein (EGFP). For flow cytometry-based assays, the optimal cell confluence was determined as 90%, and the virus was titrated using the assay. The established FCN assay follows the percentage law and an optimal MOI of not less than 5 × 10 was determined by using a purified chimeric antibody. In addition, comparison of the anti-HAdV-3 NAb titers of 72 human serum samples by the MN and FCN assays, showed that both assays correlated strongly with each other. Our FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and robotically. Importantly, the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture supernatants. Thus, this sensitive and high-throughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAdV-3 and for screening anti-HAdV-3 NAbs in cell culture supernatants.
Topics: Adenoviruses, Human; Animals; Antibodies, Neutralizing; Antibodies, Viral; CHO Cells; Cricetinae; Cricetulus; Flow Cytometry; High-Throughput Screening Assays; Mice; Neutralization Tests
PubMed: 32990935
DOI: 10.1007/s12250-020-00295-2 -
Journal of Clinical Microbiology Jan 2021Surrogate neutralization assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be done without biosafety level 3 containment and in multiple...
Surrogate neutralization assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be done without biosafety level 3 containment and in multiple species are desirable. We evaluate a recently developed surrogate virus neutralization test (sVNT) in comparison to 90% plaque reduction neutralization tests (PRNT) in human, canine, cat, and hamster sera. With PRNT as the reference, sVNT had sensitivity of 98.9% and specificity of 98.8%. Using a panel of immune sera corresponding to other coronaviruses, we confirm the lack of cross-reactivity to other coronaviruses in SARS-CoV-2 sVNT and PRNT, except for cross-reactivity to SARS-CoV-1 in sVNT.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; COVID-19 Serological Testing; Cats; Cricetinae; Cross Reactions; Dogs; Female; Humans; Immune Sera; Male; Neutralization Tests; SARS-CoV-2; Sensitivity and Specificity
PubMed: 33139421
DOI: 10.1128/JCM.02504-20 -
STAR Protocols Dec 2022Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses...
Evaluating the neutralizing antibody titer following SARS-CoV-2 vaccination is essential in defining correlates of protection. We describe an assay that uses single-cycle vesicular stomatitis virus (VSV) pseudoviruses linking a fluorophore with a spike (S) from a variant of concern (VOC). Using two fluorophores linked to two VOC S, respectively, allows us to determine the neutralization titer against two VOCs in a single run. This is a generalizable approach that saves time, samples, and run-to-run variability. For complete details on the use and execution of this protocol, please refer to Sievers et al. (2022)..
Topics: Humans; SARS-CoV-2; COVID-19; COVID-19 Vaccines; Neutralization Tests; Antibodies, Viral
PubMed: 36595901
DOI: 10.1016/j.xpro.2022.101835 -
The Indian Journal of Medical Research May 2021Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed...
BACKGROUND & OBJECTIVES
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations.
METHODS
Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous β-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test.
RESULTS
Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest.
INTERPRETATION & CONCLUSIONS
Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.
Topics: Antibodies, Viral; COVID-19; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Immunoglobulin M; Neutralization Tests; SARS-CoV-2; Sensitivity and Specificity
PubMed: 34145085
DOI: 10.4103/ijmr.IJMR_3806_20